Summary: Our laboratory studies signal transduction in the immune system with a focus on tyrosine phosphorylation-based signaling pathways. These pathways are required for intracellular signaling involved in normal cellular growth and differentiation but are also major pathways that can cause abnormal growth, differentiation and development in multiple human disorders including primary immunodeficiencies, autoimmunity and cancer. Using interdisciplinary approaches of genetics, genomics, protein biochemistry and cell biology, our goals are to understand how signaling molecules and pathways contribute to normal function of cells in the immune system and their dysfunction in disease, and how manipulation of signaling pathways can aid in the development of therapeutics. We are particularly interested in how these pathways affect responses to infectious diseases and immunization. Research:&#8232;I. Tec Kinases: Over the last 14 years, we have studied members of the Tec family tyrosine kinases. Mutations affecting the prototypical member, Btk, are responsible for the human genetic disorder X-linked Agammmaglobulimemia, characterized by abnormal B cell development and function. We have focused on the Tec members expressed in T cells, Itk and Rlk. Confirming the importance of these kinases, recent reports have described a profound lethal immunodeficiency associated with Itk mutations; polymorphisms in the Itk promoter are also associated with increased risk for asthma and atopy.&#8232; We have shown that mutations of Itk and Rlk impair T lymphocyte development and function in mice, altering responses to infections and allergic challenges. In addition to helping describe their roles in T cell receptor induced activation of PLC-g and Ca++ mobilization, we have shown that Tec kinases help regulate the actin cytoskeleton and cell adhesion, which are critical for T cells to exert effector cell functions. We have further shown that mutations of these kinases affect patterns of cytokine production by CD4+ T helper lymphocytes and the ability of mice to respond to distinct types of infectious diseases. &#8232;In work carried out in this period, we have examined the role of Tec kinases in regulating the balance between Th17 cells, an effector T cell subclass important for responses to extracellular bacteria (and which contribute to autoimmune and inflammatory disorders, including asthma), and regulatory T cells, a subset of cells responsible for keeping immune responses in check. A new focus is on the roles of Tec kinases and TCR signaling in metabolic regulation of differentiation. We are also evaluating the potential role of Itk in cytolytic responses to viral infection, given the description of Itk deficiency in patients with fatal infections with Epstein-Barr Virus (EBV). II. SAP: As an extension of these studies, we are studying other signaling molecules involved in T helper cell differentiation, including SAP, a small SH2 containing adaptor that binds phosphorylated tyrosine residues, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1), characterized by fatal infectious mononucleosis, B cell lymphomas, and antibody defects. SAP binds to the intracellular tails of the SLAM family of co-stimulatory receptors. We previously generated SAP-deficient mice and found that upon infectious challenge, these mice recapitulated features of XLP, including increased T cell activation and decreased antibody production and have demonstrated that SAP-deficient T cells fail to provide essential signals to B cells for generating germinal centers and long-term antibody responses, which are required for successful vaccination. We further found that SAP-deficient T cells have a selective defect in adhesion to B cells but not other antigen-presenting cells, preventing them from delivering the contact-dependent signals required for B cells to form germinal centers (Qi et al, Nature, 2008; Cannons et al, Immunity, 2010). Our results provided the basis for confirmatory studies showing similar defects in in XLP patients. Moreover, it suggested potential insight into the B-cell centric phenotypes in XLP, including defective T cell help for B cell antibody responses and an inability of T cells and NK cells to kill EBV-infected B cells (Schwartzberg, Nat Rev. Immunol 2009). In the last year, we have focused on two major areas. The first is to evaluate whether defective T:B cell interactions do contribute to abnormal responses to EBV-infected B cells. We have found that SAP-deficient CD8 cytotoxic lymphocytes show selective defects in interacting with and killing B cell targets. We have further linked this to altered structure of the immunological synapse, an organized structure that forms at the contact site between T cells and their targets and showed that in the absence of SAP, the negative signaling molecule SHP-1, gets recruited to the SLAM family members Ly108 and 2B4, leading to a strong negative signal that inhibits cell interactions and cytolysis. This negative signal can be blocked by SHP-1 inhibitors. Importantly, this work showed that the phenotypes of SAP-deficiency require the upstream SLAM family members that deliver a negative signal, providing insight into potential therapeutic approaches to this disease. This work, which was recently published (Zhao et al 2012, Immunity) provided important insight into XLP and the unique sensitivity to EBV, a B-cell tropic virus. Furthermore, we found similar results in CD4 cells (Kageyama et al, 2012 Immunity) providing common mechanistic insight into the pathophysiology of XLP. III. Tfh cells: A third area focuses on the role of SAP in the development and function of Tfh cells, the critical helper T cell population required for providing signals to B cells for germinal center formation. We have now defined in vitro conditions for the differentiation of a Tfh-like cell population that can rescue defects in SAP-deficient mice and have used these cells to help define requirements for Tfh cell differentiation. We have further used cellular, genetic and epigenetic studies, including ChIP-Seq to help define the cross-regulation of Tfh cells with other T cell lineages both using in vitro-derived Tfh-like, as well as ex vivo Tfh cells (Lu et al, Immunity 2011, and Nakayamada et al 2011, Immunity). Our work supports the idea of plasticity between T helper cell populations that can allow an organism to respond appropriately to distinct infectious organisms and vaccines. Understanding the cellular interactions and signals defective in SAP-deficient mice is therefore of high importance not only for understanding the pathophysiology of XLP but also for the defining the requirements for Tfh cell differentiation and the development of successful antibody-mediated immunity. IV. Our work on SAP has helped define the importance of SLAM family members in immunity. We have defined two SLAM members that have important roles in germinal center formation: CD84 and Ly108 (Cannons et al, Immunity 2010; Kageyama et al 2012, Immunity). Ly108 is of particular interest, given its implication in negative signaling (Zhao et al, Immunity 2012; Kageyama et al 2012, Immunity) as well as autoimmune susceptibility and the formation of innate T lymphocytes. We have further evaluated Ly108 and have evidence for a novel form of this receptor that is expressed only in the Lupus-resistant C57Bl/6 strain and exhibits distinct biochemical properties (Dutta et al 2012, J. Immunol). This work provides an important step in understanding biochemical properties of this potentially clinically important receptor.